Antibodies are biologically and commercially significant polypeptides that bind with great specificity and affinity to a particular target molecule, called an antigen. Antibodies are produced by immune cells of vertebrate animals, and all naturally-occurring antibodies share the same basic structure, namely two identical heavy chains covalently bonded to two identical light chains. The N-terminal regions of a single heavy chain and a single light chain form an antigen-binding site that is particular to each individual antibody. The C-terminal region of the heavy chains determines the particular isotype of the antibody, and the same antibody-producing cell can produce antibodies of different isotypes, where all the antibodies produced by the cell have the same antigen-binding site. The different isotypes typically perform different functions in the animal. For example, antibodies of the E isotype (i.e., IgE antibodies) are involved in the allergic response while antibodies of the A isotype (i.e., in IgA antibodies) can be found in mucosal membrane, saliva, and breast milk. The four-chain antibody molecule can exist by itself (e.g., an IgG antibody) or with additional monomers to form dimers (e.g., an IgA antibody) or even pentamers (e.g., an IgM antibody).
With the basic structure of an antibody well-understood, one can produce recombinant antibodies by manipulating the different regions of an antibody using standard molecular biology techniques. For example, U.S. Pat. Nos. 6,180,370 and 6,548,640 (herein incorporated by reference in their entirety) describe humanizing an antibody that naturally occurs in a non-human animal by manipulating various regions of the non-human antibody using molecular biology techniques. Other methods for manipulating or generating recombinant antibodies using standard molecular biology techniques are described (see, e.g., PCT Publication No. WO91/17271, PCT Publication No. WO92/01047; U.S. Pat. Nos. 5,969,108, 6,331,415, 7,498,024, and 7,485,291, all of which are herein incorporated by reference in their entirety).
During an immune response, an animal will generate numerous different antibodies, each with a different antigen-binding specificity. This population of antibodies is called a polyclonal population of antibodies. If the immune response is directed toward a particular antigen, most (but not all) of the polyclonal antibodies made by the animal will specifically bind that antigen. However, with differences in binding affinity and binding sites on the antigen, some of the polyclonal antibodies are more favored than other polyclonal antibodies. In their Nobel Prize-winning discovery in 1975, Kohler and Milstein discovered a way to isolate and immortalize a single antibody-producing cell, which produces a monoclonal antibody that specifically binds to the antigen of interest, from a polyclonal antibody-producing animal (Kohler and Milstein, Nature 256: 495-497, 1975). This immortalization technology, which involves fusing the antibody-producing cells to an immortalized cell to produce a monoclonal antibody-producing hybridoma, has been the industry standard for making monoclonal antibodies for the past 35 years.
Despite its popularity and its longevity, the Kohler and Milstein hybridoma method has numerous drawbacks. For example, it is very time-consuming and labor-intensive. More relevantly, given how time-consuming and labor-intensive it is, only a small fraction of the antibody-producing cells of the animal are immortalized and screened for their ability to produce an antibody that specifically binds to the antigen. Finally, even once a hybridoma with the desired antigen specificity is isolated, obtaining the amino acid sequence of the antibody to facilitate further manipulation, such as humanization, of the antibody, is arduous and time-consuming.
There is a need for improved methods for creating monoclonal antibodies that specifically bind to a desired antigen.